B adrenergic receptor kinase C-terminal peptide gene-therapy improves β2-adrenergic receptor-dependent neoangiogenesis after hindlimb ischemias

Abstract

After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of β2- adrenergic receptor (β2AR) signaling, leading to reduced revascularization. Indeed, in vivo β2AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G proteincoupled receptor kinase 2 (GRK2) is a key regulator of bAR signaling, and b adrenergic receptor kinase C-terminal peptide (bARKct), a peptide inhibitor of GRK2, has been shown to prevent bAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of β2AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated bARKct gene transfer in an experimental model of HI and its effects on bAR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant bAR down-regulation that was paralleled by an approximately 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the bARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and bAR density in the ischemic muscle, compared with control groups. The effect of bARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the bARKct fenoterol, a β2AR-agonist, induced enhanced β2AR proangiogenic signaling and increased EC function. Our results suggest that bARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced β2AR downregulation.